Our long-term OBJECTIVES remain to apply the fundamental concepts and broad technologies of cell and molecular biology to understand hepatic epithelial cell function and dysfunction. We now focus principally on cholangiocytes, epithelial cells that line intrahepatic bile ducts, because of their biologic and clinical importance, the new hypotheses and techniques we have developed for their study, and the role we have played in advancing cholangiocyte pathobiology, an underserved area of research. We will utilize novel experimental models, methods, and probes to investigate cholangiocyte water and solute transport by testing the CENTRAL HYPOTHESIS that cholangiocyte bile production is the net result of solute-driven, bi- directional, passive movement of water molecules through water- selective channels [Aquaporins (AQPs)] constituitively expressed on or recycled among cholangiocyte cellular compartments. SPECIFIC AIMS are to test the hypotheses that ductal bile formation: (i) depends on expression and cellular compartmentalization of multiple cholangiocyte AQPs; (ii) is regulated by hormone-responsive, exocytic/endocytic recycling of AQP1 to the apical cholangiocyte membrane via specific cellular tracks (actin and tubulin cytoskeleton) and molecular motors (kinesin, dynein, and dynamin); and (iii) is driven by influx (absorptive) and efflux (secretory) osmotic gradients established by solute transporters/exchangers (bile acids/glucose/ions) heterogeneously expressed in cholangiocytes. We will test these hypotheses with an array of new methods including AQP knockout mice; cultured rat cholangiocytes transfected with AQP-green fluorescent fusion constructs; immunoisolation of AQP1-containing transport vesicles; apical and basolateral cholangiocyte membrane vesicles; intact closed or perfused rat bile duct units; subpopulations of rat cholangiocytes isolated from different bile duct segments; and computer generated 3-D reconstruction of the rat biliary tree. Results of these experiments will clarify which AQPs are expressed in cholangiocytes, their intracellular topography and segmental ductal distribution, what molecules control their cellular compartmentalization, and their physiologic relevance to ductal bile formation. Innovative aspects of the program include novel methodologies and concepts regarding ductal bile formation and cholangiocyte heterogeneity. The information generated will provide a theoretical framework for development of novel therapeutic strategies for the cholangiopathies, a group of cholestatic genetic/acquired hepatobiliary diseases in which the cholangiocyte is the principal target of diverse destructive processes.